Hi all. I'm preparing qPCR standards for one of the genes I'm interested in looking at and prepared a serial dilution of it (from 10^6 to 10^1 copy number per µl). I used the standard protocol for SYBR Green assay in Stepone Plus instrument (2-step PCR; 95C for 10min followed by 40 cycles of (95C for 15s; 60C for 1 min) plus melting curve). Reaction mixture was 20µl with final primer conc. of 200 nM. cDNA template was prepared from RT-PCR and standards were prepared after confirming the primer specificity on gel and that the amplicon contains no secondary products. The standard curves align very well (r^2 is 0.998, slope is -3.038). Could not detect any secondary products in melt curve either. But what is bugging me is why does my amplification curve look so weird. Any help or suggestion is very well appreciated :). Thank you in advance and have a goo day :)
Well, both illumina and applied biosystems agree that sigmoidal amplification curve shape is related to incorrect baseline settings. This can be easily be adjusted in the program
http://support.illumina.com/real_time_pcr/nupcr/troubleshooting.html
http://www3.appliedbiosystems.com/WebTroubleshooting/AbnormalAmplificationCurves/Your_amplification_curves_have_a_sigmoidal_shape.htm
Well, both illumina and applied biosystems agree that sigmoidal amplification curve shape is related to incorrect baseline settings. This can be easily be adjusted in the program
http://support.illumina.com/real_time_pcr/nupcr/troubleshooting.html
http://www3.appliedbiosystems.com/WebTroubleshooting/AbnormalAmplificationCurves/Your_amplification_curves_have_a_sigmoidal_shape.htm
Hi Jörg: thank you for your reply. I used sequence specific primers, so I'm not sure what you meant by probes. Also, I did not use DTT in my RT-PCR. The slope is not a good one, I agree and that is what made me wonder what went wrong with the amplification. I should maybe check the baseline as you and Dovile have suggested :)
Hi Dovile: thank you for your response. I shall have a look at links you sent :)
Hi Shreyas,
Sigmoidal curves tend to occur when the template concentration is too high and/or the GOI is very abundant (16S rRNA, for example) and the automatic baseline is then not set correctly.
As far as I remember, there was an option in StepOne to change from Log to Linear view, there you would need to unclick the automatic baseline settings and adjust them manually so that the fluorescence signal is at flat zero for all of the assays and the stop cycle of the baseline is two cycles before the earliest assay emerges.
Hope this helps!
Hi, sigmoidal shape usually related with too low baseline setting in your instrument’s data analysis software....you can refit your instrument data analysis software and exclude the noise, i think it will improve your results....by the way which instrument you used...........
You can perform a baseline subtraction through resetting the software after your result appeared, your primer is specific its very clear from melting curve..
It seems that the baseline correction is not working perfectly. The signal shows a linear trend (that looks logarithmic in your plot, because the y-axis has a log scale) that is not depending on the production or accumulation of PCR products*. This trend should be removed from the amplification curves. There are several algorithms proposed to estimate and remove these trends, and your software should actually offer at least one of these methods (look for "background correction" or something similar).
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* there is no good explanation, but it may likely be the generation of eximers of the fluorophors or of some other ingredient of the reaction mixture. This effect is dependent on the buffer conditions, the ingredients, the temperature, and the exitation. It may thus be more pronounces in one assay and less in another. In any case, this effect can't be completely avoided experimentally but it can be quite easily corrected for analytically.
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